Team:HokkaidoU Japan/Notebook/aggregation Week 4
From 2012.igem.org
July 23th
Plasmid extraction
mini-prep for Ag43(7/17 cultivated colony resuspended and 7/20 cultivated colony resuspended). We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.
July 24th
Electrophoresis
Electrophoresis for Ag43(mini-preped above) and Ag43 digestion results(digested with EcoRI and SpeI)
From this digestion result, we knew that one or two enzymes didn't work successfully but there are enough concentration of DNA in 3000bp band to use in digestion.
Gel extraction
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
Ethanol precipitation
Ethanol precipitation for digestion and gel extraction product.
- Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
- Centrifuged at 15000 rpm, 10min at 4C.
- Remove supernatant and added 220 ul of 70% ethanol.
- Centrifuged at 15000 rpm, 10 min at 4C.
- Remove supernatant and air drying at room temperature then added 10 ul of DW.
From this result, we estimated that the concentration of ethanol precipitation product is about 40ng/ul.
Digestion
Digestion to confirm how many PstI cutting sites are in K346007(Ag43 coading) and Ag43-dT complex digestion with SpeI and XbaI. Ag43 PstI
DNA solution | 5 ul |
PstI | 1 ul |
10xH buffer | 2 ul |
DW | 12 ul |
Total | 20 ul |
Ag43-dT
SpeI and XbaI
DNA solution | 12 ul |
SpeI | 1 ul |
XbaI | 1 ul |
10xH buffer | 2 ul |
DW | 4 ul |
Total | 20 ul |
Electrophoresis
Electrophoresis for digestion results.
From this result, we found that there are 6 PstI cutting sites in K346007(Ag43).
Gel extraction
Gel ectraction of Ag43-dt digestio result.We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
Digestion
Digestion for Ag43-dT and pSB1AK3 mixture(each DNA fragment is about 3kbp) with HindIII to digest pSB1AK3 into about two 1.5kbp fragments.
DNA solution | 8 ul |
HindIII | 1 ul |
10xM buffer | 1 ul |
Total | 10 ul |
July 25th
Digestion
Digestion of pT7-RBS on pSB1K3 cutting with SpeI.
DNA solution | 3 ul |
SpeI | 1 ul |
10xH buffer | 1 ul |
DW | 5 ul |
Total | 10 ul |
We were confirmed that pAB1AK3 was digested into 1.3k and 1.8k bp fragments by HindIII. But there are a little doubt SpeI wasn't work because the band of pT7-RBS on pSB1K3 of d- and d+ existed same bp area.
Gel extraction
Gel extraction of Ag43-dT on pSB1AK3(HindIII) and pT7-RBS on pSB1K3(SpeI). We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
Ethanol precipitation
Ethanol precipitation of digestion and gel extraction products.
- Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
- Centrifuged at 15000 rpm, 10min at 4C.
- Remove supernatant and added 220 ul of 70% ethanol.
- Centrifuged at 15000 rpm, 5 min at 4C.
- Remove supernatant and air drying at room temperature then added 5 ul of DW.
Ligation
Ligation for pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)
pT7-RBS on pSB1K3 | 2 ul |
Ag43-dT | 2 ul |
DW | 1 ul |
Ligation Mighty Mix(TAKARA) | 5 ul |
Total | 10 ul |
Degree | Minute |
16 | 30 |
65 | 10 |
4 | Hold |
July 26th
Transformation
Transformation of plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(BL21).
- Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
- Incubated on ice for 30 min.
- Added 200 ul of LB then incubated the cells for 2 hrs at 37C.
- Prepared and Labeled two petri dishes with LBK.
- Plate 200 ul of the transformation onto first dish and spread.
- Added 450 ul of LB to 50 ul of the transformation and plated 200 ul of it onto second dish and spread.
- Incubated the plates at 37C for over 30 hrs.
Electrophoresis
Electrophoresis of digestion and ligation products.
- Placed TBE agarose gel in Electrophoresis chamber.
- Added 1/2X TBE buffer to Electrophoresis chamber.
- Added 5 ul of Etbr and ran at 100 V for 30 min.
- Load 1kb DNA ladder and each samples.
- Ran at 100 V for 30 min.
There are no band in the lane of ligation products. But if digestion products didn't ligate, there are two bands of digestion products would exist in the lane.
July 27th
Transformation
Transformation of plasmid DNA ligated at 25th (pT7-RBS-Ag43-dT on pSB1K3) in E. coli(BL21).
- Added 2 ul of plasmid DNA to 50 ul of thawed competent cells on ice.
- Incubated on ice for 30 min.
- Added 600 ul of LB then incubated the cells for 2 hrs at 37C.
- Prepared and Labeled three petri dishes with LBK X2 and LBC.
- Plate 300 ul of the transformation onto LBK dish and spread.
- Added 900 ul of LB to 100 ul of the transformation and plated 300 ul of it onto LBC dish and LBK dish then spread.
- Incubated the plates at 37C for 17 hrs and 30 min.
Ligation
Ligation of pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)
pT7-RBS on pSB1K3 | 2 ul |
Ag43-dT | 2 ul |
DW | 1 ul |
Ligation Mighty Mix(TAKARA) | 5 ul |
Total | 10 ul |
Degree | Minute |
16 | 30 |
65 | 10 |
4 | Hold |
July 28th
There were many colonies on LBC! We guess we mistook pT7-RBS on pSB1C3 for pT7-RBS on pSB1K3.
Liquid culture
Liquid culture for pT7-RBS-Ag43-dT on pSB1C3.
- Added 2 ml of LBC into culture tubes.
- Resuspended 5 colonies.
- Incubated the tubes at 30C for 22 hrs.
Digestion
Digestion of pT7-RBS on pSB1K3 with SpeI and Ag43-dT on pSB1AK3(cut with SpeI & XbaI) with HindIII. pT7-RBS on pSB1K3
DNA solution | 3 ul |
SpeI | 1 ul |
10xH buffer | 1 ul |
DW | 5 ul |
Total | 10 ul |
Ag43-dT on pSB1AK3(cut with SpeI & XbaI)
DNA solution | 8 ul |
HindIII | 1 ul |
10xM buffer | 1 ul |
Total | 10 ul |
Digestioned at 37C for 2hrs.
This results confirmed that pSB1AK3 was successfully digested into fragments(1.3K and 1.8K bp), but we weren't confirmed whether pT7-RBS on pSB1K3 was digested or not.
Gel extraction
Gel extraction for digestion production. We used FastGene Gel&PCR extraction kit(NipponGenetics)and got 50 ul of DNA solution.
Ethanol precipitation
Ethanol precipitation for digestion and gel extraction product.
- Added 5 ul of NaoAc, 1.5 ul of glycogen and 125 ul of 100% ethanol.
- Centrifuged at 15000 rpm, 10 min at 4C.
- Remove supernatant and added 220 ul of 70% ethanol.
- Centrifuged at 15000 rpm, 10 min at 4C.
- Remove supernatant and air drying at room temperature then added 10 ul of DW.
Ligation
Ligation for pT7-RBS on pSB1K3(Vector) and Ag43-dT(Insert)
pT7-RBS on pSB1K3 | 2 ul |
Ag43-dT | 2 ul |
DW | 1 ul |
Ligation Mighty Mix(TAKARA) | 5 ul |
Total | 10 ul |
Degree | Minute |
16 | 30 |
65 | 10 |
4 | Hold |
July 29th
Plusmid extraction
mini-prep for five pT7-RBS-Ag43-dT. We used FastGene Plasmid Mini Kit(Nippon Genetics) and got 50 ul of DNA solutions.
Digestion
We need to check the DNA is really pT7-RBS-Ag43-dT or not. mini-prep products(pT7-RBS-Ag43-dT) PstI
DNA solution | 4 ul |
PstI | 1 ul |
10xH buffer | 2 ul |
DW | 13 ul |
Total | 20 ul |
mini-prep products(pT7-RBS-Ag43-dT)
EcoR1 and Spe1
DNA solution | 4 ul |
EcoR1 | 1 ul |
Spe1 | 1 ul |
10xH buffer | 2 ul |
DW | 12 ul |
Total | 20 ul |
Electrophoresis
Electrophoresis for digestion results.
I could not understand what is happend. I tried it again, but the result was the same.